The general aims of the proposed research are to define the molecular mechanisms for the regulation of cyclic nucleotide metabolism in normal mammalian tissues. Specific emphasis will be placed on the identification, characterization, regulation and physiological roles for each member of the cGMP-stimulated and Ca++/calmodulin-dependent families of cyclic nucleotide phosphodiesterase. Four major approaches will be emphasized. First, full length cDNAs for each different isozyme will be isolated, characterized and expressed. Secondly, the domain organizations and interactions will be dissected using mutagenesis, deletion analysis, and chimeric PDE constructs. Thirdly, physiological function and regulation by phosphorylation and by hormones will be studied in both intact cells and in in vitro. Finally, two promising new techniques for isolation of cDNAs for novel cyclic nucleotide phosphodiesterases will be explored.